Crater formation and endocytosis in human endothelial cells in vitro.

The formation of craters under in vitro hypoxic conditions in the human venous endothelium was studied by scanning-(SEM) and transmission electron microscopy (TEM). Craters are a result of collapsed intracytoplasmatic vesicles which is probably due to extraction of fluid during processing. Thus craters are thought to be artefacts of preparation. This investigation indicates that fusion of caveolae is involved in intracytoplasmatic vesicle formation. A new quantitative method is described for studying endocytosis. Whole vein wall preparations immersed in isotonic saline (approximately 1 h) revealed reduced endocytotic activity in the luminal plasma membrane (LPM) of endothelial cells compared to the basal plasma membrane (BPM), where the endocytotic activity was increased.

Cardiac hypothermia evaluated by ultrastructural studies in man.

Transmural left ventricular biopsies from 13 patients undergoing cardiopulmonary bypass were studied. The hypertrophic myocardium was protected by deep hypothermia (15 degrees C.) during ischemic arrest for a maximal period of 96 minutes. Biopsies were taken at the start of bypass, at the end of aortic cross-clamping, and after 20 minutes of reperfusion. The structure of the mitochondria remained normal, whereas cellular alterations in the form of widened intercalated discs, interstitial and intracellular edema, myelin figures, and slight myofibrillar lysis were observed in all stages. As no clear correlation between these structural injuries and aortic cross-clamping time was seen, they are a result of cardiac hypertrophy rather than intraoperative ischemia.

New temporary atrial and ventricular pacing leads for patients after cardiac operations.

We have studied two new temporary pacing leads (Medtronic 6491 and 6492) intended for pacing after cardiac operations. The conductor has stainless steel strands coated with polyethylene connected to a 4' mm2 surface area, stainless steel, smooth, tapered electrode. A soft 4-0 coiled polypropylene fiber served as as fixation mechanism in the heart. The study included 15 children (aged 3 months to 7 years, body weight 4.4 to 20 kg) with a variety of congenital heart defects and 15 adults (aged 45 to 78 years) with coronary artery disease (n = 13) and aortic valve disease (n = 2). A pair of leads each was placed in a bipolar fashion in the right atrial wall and nonsystemic ventricle in the children (median implant duration 12 days) and in the right atrial wall only in the adults (median implant duration 9 days). The atrial current threshold values in children increased from 0.61 +/- 0.34 mA immediately after implant to 2.08 +/- 1.86 mA at explant (p < 0.002). In the adults the threshold values increased from 0.95 +/- 1.44 mA immediately after implant to 2.76 +/- 2.76 mA at explant (p < 0.002). In the ventricle the threshold values increased from 0.38 +/- 0.13 mA immediately after implant to 2.22 +/- 1.63 mA at explant (p < 0.002). Tissue resistance immediately after implant measured 809 +/- 182 omega at explant (children, p = not significant). Corresponding values in adults were 778 +/- 190 omega and 599 +/- 91 omega (p < 0.004). In the ventricle resistances changed from 1019 +/- 143 omega to 876 +/- 137 omega (p < 0.05). P wave amplitudes measured 1.8 +/- 1.5 mV immediately after implant and decreased to 1.6 +/- 1.2 mV at explant (p = not significant, children) and 2.0 +/- 1.3 mV to 1.8 mV (p = not significant, adults). R wave amplitude were 13.1 +/- 3.0 mV immediately after implant and fell to 8.7 +/- 4.5 mV at explant (p < 0.005). Thus, threshold values, tissue resistances, and electrogram and pliable amplitudes assured a safe pacemaker function. The small diameter and pliable texture of these leads provided a smooth surgical handling. They were found particularly suitable in children.

New temporary pacing lead for use after cardiac operations.

A newly designed temporary pacemaker lead (Medtronic Model 6400), which has a solid defined electrode surface area of 7.5 mm2, was compared with a multifilamental stainless steel electrode in common use (Davis & Geck, DG). Thirty-nine patients had both types of electrodes inserted intramyocardially in the right ventricle. A DG electrode fastened to the pericardium served as a reference lead. In addition to the standard Medtronic 7.5 mm2 electrode (n = 11), specially made Medtronic electrodes with areas of 5 mm2 (n = 10), 10 mm2 (n = 10), or 7.5 mm2 with platinum-iridium tips (n = 8) were studied. Measurements of myocardial stimulation threshold and resistance were made in both electrodes throughout the postoperative period. The patients' electrograms were recorded on magnetic tape for computer analysis of amplitudes and slew rates. The Medtronic 7.5 mm2 electrodes showed overall better results than 5 and 10 mm2 leads. Maximum stimulation threshold on the Medtronic 7.5 mm2 electrodes was medium 3.4 mA versus 10 mA on DG electrodes (p less than 0.001). The tissue resistance on Medtronic 7.5 mm2 was almost double than on the Davis & Geck electrodes (median 311 ohms versus 164 ohms on the day of minimum resistance, p less than 0.001). There were no significant differences in electrogram amplitudes between the two electrode types studied (5.52 versus 4.68 mV, p greater than 0.1), but the slew rates were significantly higher on the Medtronic (0.56 versus 0.37 V/sec; p less than 0.01). The new lead is an important innovation in temporary pacemaker lead design compared to the commonly used multifilamental leads.

Bipolar atrial application of a new temporary pacing lead after cardiac operations.

A new temporary pacing lead. Medtronic Model 6400, with a defined electrode surface area of 7.5 mm2, has been clinically and electrophysiologically evaluated during bipolar atrial application in 20 patients after cardiac operations. A silicon disc was used for fixation of the electrodes to the right atrium in 10 patients, and an atrial plication technique was used in the other 10. Myocardial stimulation threshold and resistance were measured throughout the postoperative period. Atrial electrograms were recorded on magnetic tape for computer analysis of amplitudes, slew rates, and signal source impedance. No significant differences (p greater than 0.1) were found in myocardial stimulation threshold between the two fixation modes. During constant-current pacing, median threshold rose from 0.65 mA to 2.3 mA. Stimulation resistance, measured during constant-voltage pacing, fell from 567 to 365 omega, with a subsequent rise to 425 omega before electrode removal. Again no difference was found between silicon disc and plication fixation of the electrodes. P-wave amplitudes were significantly higher with plication than with silicon disc fixation (2.26 versus 0.86 mV, p less than 0.01), as were slew rates (0.34 versus 0.18 V/s, p less than 0.05). Signal source impedance had a magnitude of 6 k omega. The electrodes were used successfully in 12 (60%) of the patients for diagnosis and/or treatment of arrhythmias. We find the new lead well suited for atrial application.

Primary 9 + 0 cilia in the embryonic and the adult human heart.

Primary 9 + 0 cilia have been found in the embryonic and the adult human heart. Proximally the cilia show the typical 9 + 0 filament arrangement. Rearrangement of the filaments occur in their distal regions. The cilia are usually found in deep invaginations of the cell membrane, but can also be found in a superficial position. Close connections are frequently found between cilia and thin cytoplasmic extensions from neighbouring cells.

Ultrastructural studies on the formation of myoflilaments and myofibrils in the human embryonic and adult hypertrophied heart.

The myofibrillogenesis in the human embryonic heart is described. The synthesis of thin filaments, which are the first to appear, takes place in close proximity to smooth surfaced SR tubules. Z-band material is closely related to the thin filaments and appears first as irregularly distributed patches in the filamenteous mass. Further cellular differentiation includes an organization of the thin filaments/Z-band material. The synthesis of thick filaments, which follows that of the thin filaments, takes place in ribosome rich areas of the cell. They are rapidly incorporated into the strings of organized thin filaments/Z-band material. The periodic binding sites on both kinds of filaments are believed to play an important role in the precise ordering of the filaments. The formation of myofilaments in the adult hypertrophied human heart is also described. The similarities between this process and that observed in the embryonic heart are striking, and we believe it to be the same process.

Surgical treatment of spontaneous pneumothorax by wedge resection without pleurodesis or pleurectomy.

OBJECTIVE: Evaluation of wedge resection of the lung without pleurodesis or pleurectomy as a method of surgical treatment for spontaneous pneumothorax in terms of complications, recurrence rate and postoperative complaints. METHODS: Retrospective study of 132 operations for spontaneous pneumothorax in 120 patients (84 men and 36 women: mean age 34 years, range 14-77) performed between 1974 and 1993. The mean observation time was 84 months (range 6-229) and a 100% follow-up rate of all survivors (97%) was achieved. RESULTS: The indications for surgery were recurrent pneumothorax (52%), persisting air leak during first episode (45%), or hemothorax (3%). Perioperative findings were single bullous disease (86%), 2-3 bullae (6%), diffuse bullous disease (5%) and no bullous disease in 3% of the cases. The overall complication rate was 16% (30-day mortality 1%, reoperation for postoperative bleeding 2%, bronchopneumonia 8%, new pneumothorax during hospital stay 5%). The late recurrence rate (operated lung) was 5%. All recurrences were successfully treated by drainage (n = 3), exsufflation (n = 1) or observation only (n = 3). Reoperation was not necessary. Thirty-seven percent of the patients had postoperative complaints which they associated with the operation. CONCLUSION: Lung resection without pleurodesis or pleurectomy is a simple, safe and effective method of the surgical treatment of spontaneous pneumothorax in terms of complications and recurrence rate in patients with limited bullous disease.

A quantitative study of endothelial cell injury in aorto-coronary vein grafts.

Criteria indicating injury of endothelial cells (craters, protrusion, denudation) in saphenous veins for aorto-coronary bypass grafting have been examined and quantitated by use of light-(LM), scanning electron (SEM) and transmission electron microscopy (TEM). The specimens were fixed either by immersion or under pressure. It was shown that the conventional way of handling saphenous vein grafts prior to implantation results in serious damage of the endothelial lining. The factors responsible are presumed to be hypoxia, manual flushing and distension with isotonic saline for blood removal, control of leakage, and counteracting spasm of the graft. Even samples collected by a "no touch" technique and exposed to a short hypoxic interval sometimes revealed slight injury.

Inflammatory reaction as an occlusive mechanism in aortocoronary bypass grafts?

Morphological changes in aortocoronary vein grafts are described in two cases; one dying 5 hours post operatively and the other reoperated 8 months after aortocoronary bypass surgery for graft occlusion. Occlusive thrombi and fibrin deposition on the intimal surface and a focal inflammatory process in the wall of the vein grafts were found in the former. Thrombotic encrustation and organization similar to the manifestations of the thrombogenic theory of atherosclerosis were demonstrated in the latter.

Traumatic arteriovenous fistulas. A follow-up study.

Over the period 1958 to 1972 14 patients with traumatic arteriovenous fistulas were treated. The mean period from trauma to surgical closure of the fistula was 13.3 years. No form of reconstructive vascular surgery had been performed initially. The dominating symptom of arteriovenous fistula was a pulsating tumor with a marked thrill. Excision of the fistula and reconstruction of the artery and vein was performed in 11 patients. In 3 patients the fistula was first ligated resulting in early recurrence of the shunt. At follow-up 3-15 years postoperatively all fistulas were closed, but 5 patients had moderate symptoms.

Continuous measurement of aortic blood velocity, after cardiac surgery, by means of an extractable Doppler ultrasound probe.

A new method has been developed for the continuous measurement of aortic blood velocity in patients following cardiac surgery. Using an extractable Doppler ultrasound probe placed on the ascending aorta, the changes in aortic velocity were recorded up to 24 h postoperatively, in 14 patients undergoing coronary bypass surgery. Volume flow rate is calculated from the mean velocity, the diameter of the aorta and the angle between the ultrasound beam and the direction of the blood flow, by means of an analogue flow calculator. Estimation of aortic flow showed a correlation of r = 0.79 with cardiac output measured by a thermodilution technique. The main advantage of the system is that it allows continuous monitoring of cardiac output, as well as short and long-term trend analyses, during the early postoperative period.

Anesthesiologist-manned helicopters and regionalized extracorporeal circulation facilities: a unique chance in deep hypothermia.

Norway has nine light ambulance helicopters, four heavy sea rescue helicopters and seven ambulance air planes in 24-hours duty spread all over the country. Most are manned with anesthesiologists. Five regional hospitals in all parts of the country, offer facilities for extracorporeal circulation. A case in which a 33 year old woman was found hypothermic at 21 degrees C is presented. She developed ventricular fibrillation at the time of her rescue. She was intubated and received chest compression for 70 minutes until she was rewarmed by extracorporeal circulation. She was discharged without signs of cerebral damage. The decision to bypass less advanced hospitals en route to the regional hospital proved correct in this case, and is suggested as standard procedure in deep hypothermic patients.

Morphologic and morphometric analysis of the mast cells from human heart biopsies.

We have examined mast cell morphology in human papillary muscles obtained from patients subjected to mitral valve replacement. Elongated, metachromatically staining mast cells were found in the connective tissue close to blood vessels and nerves. Electron microscopic examination showed that the most conspicuous features were cytoplasmic granules which displayed a crystalline internal structure and which were surrounded by perigranular membranes. They were composed of a number of coiled electron-dense layers (3 to 4 predominantly) which formed scrolls and gratings. Apart from the crystalline structure, all granules had a dark, uniform matrix. Measurements of crystalline structure periodicity indicated a unimodal distribution around a mean of 85.6 +/- 1.9 A.

On the origin of Z-band material and myofilaments in myoblasts from the human atrial wall.

The origin of cardiac myofibrils in cells from the atrial wall in human embryos was studied, Z-band substance appears throughout the cytoplasm as irregular electron dense patches in a network of thin filaments. The thin and thick filaments are synthesized as separate units in the sarcoplasm and are later aggregated into myofibrils. Complexes of Z substance and thin filaments occur numerously at different stages of myofibrillar organisation. Thick filaments are formed in close proximity to free ribosomes and are later incorporated in an hexagonal pattern into the Z-band/thin filament complex.

Calcium and magnesium levels in isolated mitochondria from human cardiac biopsies.

A non-enzymatic method is presented for isolating mitochondria from small-sized human cardiac samples, including ventricular needle biopsies of 15-25 mg of wet weight. Electron microscopy demonstrates that these fractions are rich in structurally well preserved mitochondria. Calcium and magnesium levels of fractions are determined by atomic absorption flame spectroscopy. Comparative analyses are made in similar fractions of the mouse ventricle. Calcium concentrations of mitocondria isolated in the presence of ruthenium red do not differ significantly between the human auricle and ventricle, averaging 61 nmol Ca/mg protein and 68 nmol Ca/mg protein, respectively. Mitochondrial calcium level is lower in the mouse ventricular fractions, averaging 7 nmol Ca/mg protein. Mitochondrial magnesium amounts to slightly less than 60% of the calcium levels in the human heart, while it exceeds the calcium level by more than 100 per cent in the mouse heart. There is no significant difference of mitochondrial calcium between normal auricles, and, auricles of patients with increased right atrial mean pressure and/or volume overload.

The T-tubule system in the myocardia of the sand rat and mouse as demonstrated by horseradish peroxidase.

The transverse tubule (T-tubule) system in papillary muscles of the sand rat and the mouse were studied with the aid of a diffusion tracer (horseradish peroxidase). The T-tubule system in the sand rat showed a typical mammalian pattern with sarcolemmal tubules invaginating at the Z-band level of the sarcomere. These tubules follow a transverse direction in the cell with frequent longitudinal side-branches which connect tubules at different Z-band levels. In the mouse myocardium, the T-tubules also start as lateral invaginations from the sarcolemma at the Z-band levels. In the cell interior, however, the tubules ramify and brake up into a complicated system of spirally running tubules. These spirals, of relative small diameter (400A--700A), frequently expand and form lobulated cisternae at the Z-band levels.

Calcium in isolated mitochondria from the left ventricular wall.

The content of calcium per mg mitochondrial protein has been measured by conventional biochemical methods in myocardial tissue of some mammalian species. In addition, a method is presented for (1) the analysis of mitochondrial volumes in the same tissues and (2) calculating the amount of calcium in units of 10(6) mitochondria. It appears that a highly significant correlation exists between the calcium content and the number of mitochondria, with a positive correlation coefficient of 0.92. The mean mitochondrial volume in fractions of the rabbit myocardium was found to be 1.3386 micron3. Electron microscopic studies demonstrate pure mitochondrial fractions and only moderate structural alterations. The method described may represent a useful supplement for the estimation of calcium fluxes in mitochondria and of alterations in their volume, number and structure under conditions of myocardial ischemia.

Preservation of shock-frozen myocardial tissue as shown by cryo-ultramicrotomy and freeze-fracture studies.

Myocardial tissue was quickly quench-frozen by letting the specimen fall either into liquid nitrogen (LN2) or into LN2-slush, or, by drill-propelling the specimen into LN2 at a speed of 1.5 m/s. The preservation of the tissues was studied in ultrathin, dry-cut and freeze-dried cryo-sections. Shock-freezing by propelling into LN2 yields extensive areas of well preserved tissue without hole damage. Quenching into LN2-slush shows variable results, while a maximum of hole damage is obtained by letting the specimen fall into LN2. Replicas of freeze-fractured tissue which had been quench-frozen by the same methods confirmed these observations. By the drill-propelling, areas 2000-5000 micrometer2 wide reveal good preservation without visible ice crystals. Such areas occur within a superficial band of tissue to a depth of c. 20 micrometer. Even at 35 micrometer and 50 micrometer depth good preservation may be registered at the periphery of the replicas. Description is given of modifications in accessories and methods in order to make the LKB CryoKit more suitable as a routine instrument. These modifications cover replacement of the knife coolant container by a coolant brass container in open connection with three copper tubes, replacement of one of the LKB Dewar flasks by a 25 litre Dewar equipped with a Balzers filling device, and replacement of the LKB plastic sleeve fixed to the back of the specimen coolant container, by two perspex-glass discs mounted in the slit between the Ultrotome base and the cryo-chamber. In addition, modified constructions are presented of the grid-carrier, the press-assembly as well as of the container for freeze-drying and warming up of the frozen sections.